SHC and GRB2 protein signaling molecules form a complex in response to growth factor or oncogenic transformation, as described in Rozakis-Adcock et al Nature 360: 689-92 (1992). U.S. Pat. No. 6,090,621. These proteins are thought to transmit mitogenic signals from receptor and non-receptor tyrosine kinases to ras, a member of a major class of oncogenes and proto-oncogenes that encode G proteins that are located on the inner face of the plasma membrane, where they bind and hydrolyze GTP. Ras proteins are involved in an unknown way in growth-factor stimulation of cell proliferation, as described in Alberts et al., Molecular Biology of the Cell (second edition, Garland publishing New York, 1989) pp. 699 and 705. The precise mechanism of the action of ras remains unknown, as indicated in Lowenstein et al Cell 70: 431-42 (1992) and Gale et al Nature 363: 88-92 (1993).
By expression interaction cloning, the GRB2 SH3 domains were found to bind to a GRB2-associated signaling inositol polyphosphate 5-phosphatase (called SIP-110), a 110 kDa protein believed to be involved in signaling events that follow growth factor stimulation, and occur between the cell surface and transcriptional activation events. Furthermore, SIP-110 is believed to participate in modulating signaling by ras and by phosphatidyl inositol 3-kinase (PI 3-kinase), two known regulators of cell growth. It would be advantageous in the process of elucidating the mechanism of ras, PI 3-kinase and other signaling molecules and pathways, to discover other signaling molecules that participate in the signal transduction that modulates the activity of the ras pathway, the PI 3-kinase pathway, the MAP kinase pathway, the calcium signaling pathway and other signaling pathways such that cellular responses including growth and proliferation may be regulated by regulating such signaling molecules.
Activation of phosphatidylinositol 3′-kinase (PI 3-kinase) by growth factors and oncogenes has been implicated as a critical step in mitogenic signaling and cellular transformation, as described in Cantley et al, Cell 64:281-302 (1991), Kapeller and Cantley. Bioessays 16:565-76 (1994), and Stephens et al, Biochim Biophys Acta 1179:27-75 (1993), PI 3-kinase consists of 85 kDa and 110 kDa subunits which associate with receptor tyrosine kinases and intracellular signaling molecules in response to treatment with growth factors or in transformed cells. Blockade of PI 3-kinase function either by mutagenesis or with pharmacological inhibitors prevents mitogenic signaling. Further, two products of PI 3-kinase, PtdIns(3,4,5)P3 and PtdIns(3,4)P2, increase in cells treated with mitogenic stimuli as Hawkins, et al. Nature 358:157-910, (1992) and Klippel et al, Molecular and Cellular Biology 16:41174127 (1996). The products of PI 3-kinase are presumed to act as second messengers or as regulators of protein-protein interactions. The regulation of PI 3-kinase activity during signaling is less well studied.
Changes in subcellular localization, in phosphorylation state and in conformation of the enzyme have been suggested to contribute to activation but little is known about how PI 3-kinase might be down-regulated. It would be advantageous to discover and characterize molecules implicated in PI 3-kinase mediated pathways, as a means to learning how to regulate PI 3-kinase. A number of distinct forms of inositol polyphosphate 5-phosphatase have been identified. Emeaux et al., Biochim. Biophys. Acta 1436, 185-99, 1998. There is, therefore, the possibility that additional forms of this enzyme exist which can be regulated to provide therapeutic effects.